Iron is an essential metal nutrient that plays physiologically and pathologically important roles in biological systems. However, studies on the trafficking, storage, and functions of iron itself in living samples have remained challenging due to the lack of efficient methods for monitoring labile intracellular iron. Herein, we report a new class of Fe(2+)-selective fluorescent probes based on the spirocyclization of hydroxymethylrhodamine and hydroxymethylrhodol scaffolds controlled by using our recently established N-oxide chemistry as a Fe(2+)-selective switch of fluorescence response. By suppressing the background signal, the spirocyclization strategy improved the turn-on rate dramatically, and reducing the size of the substituents of the N-oxide group enhanced the reaction rate against Fe(2+), compared with the first generation N-oxide based Fe(2+) probe, RhoNox-1. These new probes showed significant enhancements in the fluorescence signal against not only the exogenously loaded Fe(2+) but also the endogenous Fe(2+) levels. Furthermore, we succeeded in monitoring the accumulation of labile iron in the lysosome induced by transferrin-mediated endocytosis with a turn-on fluorescence response.
Iron is an essential transition metal species for all living organisms and plays various physiologically important roles on the basis of its redox activity; accordingly, the disruption of iron homeostasis triggers oxidative stress and cellular damage. Therefore, cells have developed sophisticated iron-uptake machinery to acquire iron while protecting cells from uncontrolled oxidative damage during the uptake process. To examine the detailed mechanism of iron uptake while controlling the redox status, it is necessary to develop useful methods with redox state selectivity, sensitivity, and organelle specificity to monitor labile iron, which is weakly bound to subcellular ligands. Here, we report the development of Mem-RhoNox to monitor local Fe(II) at the surface of the plasma membrane of living cells. The redox state-selective fluorescence response of the probe relies on our recently developed N-oxide strategy, which is applicable to fluorophores with dialkylarylamine in their π-conjugation systems. Mem-RhoNox consists of the N-oxygenated rhodamine scaffold, which has two arms, both of which are tethered with palmitoyl groups as membrane-anchoring domains. In an aqueous buffer, Ac-RhoNox, a model compound of Mem-RhoNox, shows a fluorescence turn-on response to the Fe(II) redox state-selectively. An imaging study with Mem-RhoNox and its derivatives reveals that labile Fe(II) is transiently generated during the major iron-uptake pathways: endocytotic uptake and direct transport. Furthermore, Mem-RhoNox is capable of monitoring endosomal Fe(II) in primary cultured neurons during endocytotic uptake. This report is the first example that identifies the generation of Fe(II) over the course of cellular iron-uptake processes.
High-throughput methods
for monitoring subcellular labile Fe(II)
are important for conducting studies on iron homeostasis and for the
discovery of potential drug candidates for the treatment of iron deficiency
or overload. Herein, a highly sensitive and robust fluorescent probe
for the detection of intracellular labile Fe(II) is described. The
probe was designed through the rational optimization of the reactivity
and responsiveness for an Fe(II)-induced fluorogenic reaction based
on deoxygenation of an N-oxide, which was developed
in-house. The probe is ready to use for a 96-well-plate-based high-content
imaging of labile Fe(II) in living cells. Using this simple method,
we were able to conduct high-throughput screening of a chemical library
containing 3399 compounds. The compound lomofungin was identified
as a potential drug candidate for the intracellular enhancement of
labile Fe(II) via a novel mechanism in which the ferritin protein
was downregulated.
Mitochondria are iron-rich organelles that are involved in the process of energy production through the electron-transporting system and heme synthesis. We developed a new mitochondria-targeted fluorescent probe, MtFluNox/Ac-MtFluNox, for Fe(ii) based on N-oxide chemistry, which we recently established as a Fe(ii)-selective fluorogenic switch. The deacetylated form MtFluNox showed a turn-on response towards Fe(ii) with high metal selectivity in cuvette experiments, and an imaging study using its cell-compatible analogue Ac-MtFluNox demonstrated mitochondria-specific fluorescence enhancement in response to Fe(ii) in living cells. Furthermore, the probe was able to detect endogenously accumulated Fe(ii) induced as a result of the inhibition of heme synthesis.
Ovarian endometriosis is a recognized risk for infertility and epithelial ovarian cancer, presumably due to iron overload resulting from repeated hemorrhage. To find a clue for early detection and prevention of ovarian endometriosis-associated cancer, it is mandatory to evaluate catalytic (labile) ferrous iron (catalytic Fe(II)) and to study iron manipulation in ovarian endometriotic lesions. By the use of tissues from women of ovarian endometriosis as well as endometrial tissue from women with and without endometriosis, we for the first time performed histological analysis and cellular detection of catalytic Fe(II) with a specific fluorescent probe (HMRhoNox-M), and further evaluated iron transport proteins in the human specimens and in co-culture experiments using immortalized human eutopic/ectopic endometrial stromal cells (ESCs) in the presence or absence of epithelial cells (EpCs). The amounts of catalytic Fe(II) were higher in ectopic endometrial stromal cells (ecESCs) than in normal eutopic endometrial stromal cells (n-euESCs) both in the tissues and in the corresponding immortalized ESCs. ecESCs exhibited higher transferrin receptor 1 expression both in vivo and in vitro and lower ferroportin expression in vivo than n-euESCs, leading to sustained iron uptake. In co-culture experiments of ESCs with iron-loaded EpCs, ecESCs received catalytic ferrous iron from EpCs, but n-euESCs did not. These data suggest that ecESC play a protective role for cancer-target epithelial cells by collecting excess iron, and that these characteristics are retained in the immortalized ecESCs.
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